👉 Go to Delphi Poll Statements

1 Responders

# First_name Last_name
1 Julia Alten
2 Monika Brüggemann
3 Mireia Camós
4 Gianni Cazzaniga
5 Emmanuelle Clappier
6 Conny Eckert
7 Sarah Elitzur
8 Nicola Gökbuget
9 Csongor Kiss
10 Josep Maria Ribera
11 Nick Short
12 Dave Teachey
13 Jan Trka
14 Bela Wrench
15 Vincent van der Velden

2 Patient population and treatment protocol

2.1 Which ALL patient group do you primarily work with?

2.2 Which clinical study group do you represent?

2.3 Which treatment protocol are your patients primarily treated under during first-line therapy?

  • MD Anderson protocols - HCVAD+blina+/-INO, mini-hyper-CVD+ino+blina, blina+ponatinib
  • PETHEMA ALL2019 PETHEMA ALLPh 2022
  • GMALL Regstry, GMALL Evolve, GMALL BLIVEN
  • HOVON-100 study protocol (if unfit: according to Hovon-117/UKALL XII study protocol (unfit arm))
  • GMALL Registry younger GMALL Registry older GMALL Evolve
  • AIEOP-BFM ALL 2017, Interfant-21, EsPhALL/COG, IntReALL (SR and HR)
  • GRAALL/GRAAPH-2014 EWALL-INO EWALL-Ph03
  • AIEOP BFM 2017
  • BFM ALL-IC 2022
  • ALL Together EsPhALL for Ph+ B-ALL
  • AALL1731, AALL1732, AALL2331, AALL2321

  • first-line: AIEOP BFM ALL 2017, ALLTogether 1 second line: IntReALL 2010 HR, ALL-REZ BFM Registry, soon IntReALL BCP 2920, HemiSmart

    We only work with the second line therapy protocols.
  • INITIALL; SJALL23H; SJALL23T; Total 17
  • UKALL 14
  • AIEOP-BFM ALL 2017 (AIEOP-BFM ALL in general) EsPhALL/COG Interfant-21

3 Centralized vs. Local Processing of Samples

3.1 Is cytomorphology analysis performed centrally for your diagnostic samples?

3.2 Is cytomorphology analysis performed centrally for your follow-up samples?

3.3 Is the diagnostic immunophenotyping by flow cytometry performed centrally for your samples?

Category Comment
Other It is recommended that immunophenotyping is performed in a central lab, but it is only prtially done centrally
Other All samples are analyzed by accredited, “matured” national central laboratories.
Other Diagnostic flow is centralized in 11 reference laboratories

3.4 Is flow cytometric MRD analysis performed centrally for your samples?

Category Comment
Other MRD Flow is only done if no molecular MRD marker is available
Other All centralized except Monza center.
Other See reply to previous Q.
Other Flow-MRD is centralized in 11 reference laboratories
Other B-ALL on trial: COG approved labs. Off study local labs. Infant and T-ALL are central labs.

3.5 Is molecular MRD analysis performed centrally for your samples?

Category Comment
Other Ig/TC central, BCR::ABL partly central
Other All centralized except Padua center
Other Molecular MRD is being performed as reasearch projects by some ALLIC national team laboratories. The method is not yet available for diagnostic purposes.
Other Molecular-MRD is centralized in 2 accredited laboratories at H. Sant Joan de Deu (Barcelona) and H. Niño Jesús (Madrid)
Other Molecular MRD is performed in subsets of patients with B-ALL

3.6 Are central Flow Cytometry and Molecular MRD analyses performed in the same central laboratory or in separate laboratories?

Category Comment
Other partly overlapping
Other Flow-MRD and molecular-MRD are performed in the same lab only in the 2 molecular reference labs. For the rest of patients, samples are sent for the corresponding Flow-MRD lab and one of the two accredited molecular-MRD laboratories

3.7 If Flow Cytometry and Molecular MRD analyses are performed in two different laboratories, how is the sample handled?

Category Comment
Other several vials are taken. In theory the first sample should be send to molecular MRD (except day 15) and the second/third sample to Flow Cytometry and Moelcular genetics.

4 MRD-based risk/treatment stratification during first-line treatment

4.1 What time points are defined in your protocol for performing bone marrow aspiration for remission assessment and/or risk stratification?

  • End of Consolidation (2 times)
  • End of Induction (2 times)
  • Afrterinterphase
  • After consolidation A (younger) after consolidation I (elderly)
  • After consolidation B (younger) after remission induction II (elderly)
  • after consolidation I
  • After Consolidation I
  • after each consolidation block (at count recovery)
  • After HR-1’ when hematopoiesis has recovered (HR only)
  • After HR-2’ when hematopoiesis has recovered (HR only)
  • After HR-3’ when hematopoiesis has recovered (HR only)
  • after induction during relapse treatment
  • after intensification (at count recovery)
  • After intenstification 1A (younger) after consolidation II (elderly)
  • after phase 1 induction (at count recovery)
  • after phase 2 induction (at count recovery)
  • After remission induction I
  • After the first three HR blocks (HR1, HR2, HR3) each if CR was not achieved ealier, or previous MRD was <1% but >/= 0.1%
  • Ater intensification 1B (Younger)
  • Before Consolidation I
  • before stem cell transplantation during relapse treatment
  • D15 (mid-induction)
  • D33 (EOI)
  • D78 (end-of-early-intensification, i.e. PIB)
  • day +15
  • Day 15 (only Flow-MRD) (all risk groups)
  • Day 15 bone marrow
  • Day 15 of Induction (FCM-MRD)
  • Day 29, before consolidation 1 (all risk groups)
  • Day 33 (End of Induction IA) (PCR-MRD; FCM-MRD in case of missing Ig/TCR Targets)
  • day 33 (post induction)
  • Day 50; day 65/71/78 (according to MRD d29 & d50); post-nelarabine (day 87/92/99); post-NOPHO block A, B, C; before HSCT (HR-T-cell)
  • Day 57, day 99 (DS-HR)
  • Day 71/78, block A1; day 92, block A1; day 99, block B1; day 113, block B1, day 120, block C1; day 134, block C1; day 176, block B2; day 197, block C2, before HSCT (HR)
  • Day 78, consolidation 2 (IR-low, IR-high, DS-IR)
  • Day 78/Day 92 (End of Consolidation) (PCR-MRD; FCM-MRD in case of missing Ig/TCR Targets)
  • Day 8 peripheral blood
  • End early consolidation
  • End induction
  • End late consoloidation or Month 3 after alloHSCT
  • End of consolidation, day +252
  • end of Consolidation, day +78/+92
  • End of cycle 4, day +84
  • end of Induction, day +33
  • End of induction, day+21
  • Every 6 monys for the second year (chemotheraoy or alloHSCT)
  • Every three months after late consolidation of ater alloHSCT (1st year)
  • Mid induction
  • Some trials have additional timepoints but it varies significantly
  • week 12 (in non-SR patients)

4.2 What are the relevant cut-off values used for risk stratification?

Statement 1: MRD thresholds below 1×10⁻⁴ are not relevant for risk stratification in frontline treatment of ALL.

5 Postremission MRD monitoring for detection of an MRD relapse

5.1 In which patients do you typically perform bone marrow aspiration for an early detection of an MRD relapse?

Category Comment
Other Not systematically performed
Other before and after allo-SCT, after relapse, after CART
Other We follow the protocol. Additionally, in case of relapse suspicion based on clinical or laboratory findings
Other Before and after allo-SCT
Other all because all are relapses
Statement 2: Bone marrow aspiration should be performed in all patients for postremission MRD monitoring to enable early detection of relapse.

5.2 How many times do you perform bone marrow aspiration for relapse detection according to your protocol?

Category Comment
Other depends on a situation, can be more than four times, follow up post-SCT is usually day30, day 60, day 180, 1 yr, 2yr, 3yr
Other Never. Transplant is not included on our trials.
Other currentl not defined, very frequent
Other within the first year after each block
Other depending of the MRD-kinetics and the risk profile (3-5 x in HR without alloHSCT and 8-10 x in pts with alloHSCT)

5.3 Do you conduct additional MRD assessments based on individual clinical decisions?

5.4 At what intervals per patient do you perform post-remission MRD assessment for detection of an MRD relapse?

Category Comment
Clinical Signs prolonged cytopenias
Clinical Signs Standard risk (after TP1)/Medium risk patients (after TP2): Only if there are clincal or laboratory signs of relapse (e.g. prolonged cytopenia…)
Fixed Intervals Every 3-4 months
Fixed Intervals evry three months at 1st year, every six months for the 2nd year and then yearly up to 5 years
Fixed Intervals Ph negative ALL, chemotherapy: first year: prior to each consolidation cycle, second+third year: every 3 months, fourth year: evey 6 months. Ph negative ALL, after SCT: every 3 months. Ph+ ALL, after SCT: every 6 weeks in blood, every 3 months in BM.
Fixed Intervals every 3 months (three years)/ 2 months (2 years), and if there are clinical signs of relapse
Fixed Intervals 3
Fixed Intervals depends on a situation, follow up post-SCT is usually day 30, day 60, day 180, 1 yr, 2yr, 3yr, in relapsed based on relapse protocol
Fixed Intervals end of therapy
Fixed Intervals MRD post alloHSCT (d+30, d+60, d+100, d+180, d+365)
Other If there are clinical/lab signs suspicious for relapse, and in pts. after alloHSCT
Other after each treatment block, ad hoc practice in maintenance every 3 months
Other MRD after each element of HR-Therapy (AIEOP-BFM:all HR-patients: TPHR1, TPHR2, TPHR3 (=MRD after each HR-Block), additionally all HR-T-ALL, all Infants, B-ALL (not atl east 2 x negative in subsequent time points): prior to Re-Induction (prior to 2. Protocol III and 3. Protocol III)
Statement 3: Postremission MRD assessment should be performed at fixed intervals according to the protocol and additionally in response to clinical signs of relapse.

5.5 Until when do you perform bone marrow aspiration for relapse detection?

Category Comment
Dx Months ~36 months
Dx Months until end of year 4 (chemotherapy patients)
Dx Months uptill 5 years after reaching CR
Dx Months approximately 36-48 months
Dx Months x1 at end of therapy
Dx Months end of intensive chemotherapy (see question 16)
Transplant Months Not fully standardized. Depends on MRD kinetics.
Transplant Months approximately 36 months
Transplant Months see above, in negative samples post-SCT last BM is 3 years post-SCT, after that yearly PB. But depends on the clinical situation and individual decision of physicians
Transplant Months 1 year
Lifelong evry three months at 1st year, every six months for the 2nd year and then yearly up to 5 years
Other evry three months at 1st year, every six months for the 2nd year and then yearly up to 5 years
Statement 4: Bone marrow aspiration for relapse detection should be continued until 36–60 months after initial diagnosis.
Statement 5: Bone marrow aspiration for relapse detection should be continued until 36 months after stem cell transplantation.

5.6 What are the relevant MRD cut-off values used for relapse detection in Ph-negative ALL?

Response Answer
Other Positivity below QR: warning signal, MRD >1XE-04: molecular relapse
Other 5x10-2
Other any MRD positivity, but in positive not quantifiable verified by NGS
Other Any MRD-positivity with further confirmation with other method and different sample
Other We follow PDL guidelines
Other For relapse definition more than 1 method is needed for confirmation, if MRD is < 5%. MRD of 5E-04 is highly suspicious of pending relapse if detected late in therapy
Statement 6: For postremission relapse detection, any MRD positivity is considered relevant; however, values below 1×10⁻⁴ should be confirmed using an additional method and a second sample.

5.7 What are the relevant IG/TR-based MRD cut-off values used for relapse detection in Ph-positive ALL?

Response Answer
Other not fully standardized, probably like in Ph-negative ALL.
Other 5x10-2
Other we do BCR/ABL on DNA level simultaneously and solve individually (CML-like cases)
Other Any MRD-positivity with further confirmation with other method and different sample
Other We follow PDL guidelines
Other do not use IG/TR-MRD; 1x10-2 for flow
Other see question 18

5.8 What are the relevant BCR::ABL1-based MRD cut-off values used for relapse detection in Ph-positive ALL?

Response Answer
Other Likely not relevant with IG/TR testing
Other none
Other reported in the context of simultaneous measurement of DNA-based BCR/ABL and Ig/TR (e.g. BCR/ABL 1E-02 and Ig/TR negative does not need to be relapse in CML-like cases)
Other ALLIC group did not define MRD cut-off value for diagnosing molecular relapse in Ph+ALL. LOQ in CML is 1e-05
Other Any MRD-positivity with further confirmation with other method and different sample
Other We follow PDL guidelines
Other BCR::ABL1 positivity without Ig/TCR positivity and zytomorphological absence of blasts is not considered a relapse (this constellation is suspicious for misdiagnosis in the beginning –> CML in blasts crisis )

6 Relapse confirmation

6.1 Is a conversion to MRD-positivity considered equivalent to relapse?

Category Comment
Yes, always Yes if converts from negative to positive but only if there is high confidence that the finding reflects true MRD (e.g. not “background”), ideally confirmed on a follow-up sample.
Dependes on MRD level If ther is higher than 1x10e-4, confirmed in a second sample
Dependes on MRD level 1xE-04
Dependes on MRD level if >0,01%
Dependes on MRD level 10-4 but this is defined then as molecular relapse which is different from hematologic relapse
Dependes on MRD level Clearly if 1E-02, but it would trigger clinical actions if 1E-04 if confirmed by 2 tecniques in 2 different consecutive samples
Dependes on MRD level 1x10-2
Dependes on MRD level one log increase required
Dependes on MRD level MRD 1 -<5%: two other tests with >=1% or second BM. MRD <25% one additional test >= 1%. doi: 10.1182/blood.2021012328. PMID: 34192312; PMCID: PMC8952186.
No, never depends on ALL type-different situation in CML-like ALL, see above. In Ph-neg ALL pos NQ after negative always verified by NGS
No, never Only if MRD exceedes the 1% (1–02) limit for CR as defined by the P-d-L paper
Depends on additional findings MRD-positivity conversion should be unambiguous: either high level, or if low, without any doubt (confirmed on a second sample or a second marker or technique, or with clinical signs of extramedullary relapse))
Depends on additional findings Flow or/and morphology
Statement 7: Conversion to MRD positivity should be considered a relapse only if the MRD level exceeds 1×10⁻⁴ and is confirmed by an additional method or other molecular target.

6.2 Should the conversion from IG/TR-based MRD-negativity to MRD-positivity be confirmed by a consecutive sample?

Response Answer
Other depends on the context and MRD level
Other We follow PdL so depends on if a confirmatory test and level of detection
Other depends on the level <10-4 likely requires repeat
Other No, a single positive result is sufficient: depends on the Level (see 21)
Statement 8: Conversion from IG/TR-based MRD-negativity to MRD-positivity should be confirmed by a consecutive sample only if the MRD level is below 1×10⁻⁴.

6.3 Should the conversion from BCR::ABL1-based MRD-negativity to MRD-positivity be confirmed by a consecutive sample?

Response Answer
Other Only if accompanied by MRD by IG/TR
Other IG/TR to be analyzed
Other Relapse should be confirmed by IG/TR MRD in bone marrow
Other in CML like completely different situation, usually repeated sample necessary
Other We follow PdL
Other no routine BCR::ABL1 monitoring performed
Statement 9: Conversion from BCR::ABL1-based MRD-negativity to MRD-positivity should be considered a relapse only if accompanied by IG/TR-based MRD positivity.

6.4 If MRD conversion requires confirmation, within what time frame should the consecutive sample be taken?

Category Comment
Other No clear rule
Other 2 weeks
Other We follow PdL
Other <1 week for positive >1% without morphology, 2-4 weeks for lower level
Statement 10: If MRD conversion requires confirmation, the consecutive sample should be taken within 1–4 weeks.

7 Clinical consequences of postremission MRD reoccurence

7.1 What are the clinical consequences when MRD recurrence is detected during first-line chemotherapy?

Response Answer
Other it depends on MRD levels, frequently, a further BM is planned to monitor increase
Other No defined rule, to be discussed
Other depends on the protocol, usually SCT with salvage before
Other It depends on the MRD level, time point and the clinical setting
Other If meets PdL criteria for relapse we treat as a relapse
Other answers depend of the level of MRD positivity: >=5E-04 after TP2 (day 78/92) Indication for alloHSCT and intensification of therapy. Below 5E-4: Obersevation without immediate intervention

7.2 What are the clinical consequences when MRD recurrence is detected after the end of first-line chemotherapy?

Response Answer
Other depending on CR time, Relapse within 1 year: salvage; if CR>1 year first line induction rretarted
Other A further BM is planned to monitor increase
Other No defined rule, to be discussed
Other not measured in non-HR patients, in HR patients is defined by protocol
Other It depends on the MRD level, time point and the clinical setting
Other If meets PdL criteria for relapse we treat as a relapse
Other it depends on the MRD level see question 25

7.3 What are the clinical consequences when MRD recurrence is detected after stem cell transplantation?

Response Answer
Other not fully standardized
Other if no GVHD: reduce immune suppression. Otherwise DLI or blina
Other reduction of immunosuppression; short term confirmation of MRD increase on another BM sample
Other based on the level-low MRD IST tapering, DLI, in high level CART, 2nd SCT..
Other Individual decision made by the transplant team
Other It depends on the MRD level, time point and the clinical setting
Other Depends on type of transplant, availability of DLI, ability to add a targeted or immunotherapeutic agent
Other it depends on the MRD level. Low positivity: reduction of Immunosuppression if possible, DLI

8 Bone marrow sampling

8.1 In the clinical study your patients are treated in, is there a standard operation procedure (SOP) for bone marrow sampling?

Category Comment
Other There is a manual that states: 4.6.3 Material  10 ml Knochenmark und/oder 10 ml peripheres Blut  Von jedem Untersuchungszeitpunkt, möglichst 1. Aspirat  EDTA bevorzugt, Heparin auch möglich Die Zeitpunkte der Remissionskontrollen und Einsendung von Probenmaterial zur MRD-Untersuchung sind den aktuellen Protokollen bzw. Therapieempfehlungen zu entnehmen. Praktisch wird die KM-Punktion jeweils nach Regeneration durchgeführt. Es handelt sich also nicht um Aplasie-Punktionen. In addition, we hav a local SOP in Kiel
Other instructions are give in an extra document of the sample request form

8.2 Apart from its use in diagnosing and classifying ALL, how else is diagnostic bone marrow aspirate utilized in clinical or research settings?

Response Answer
Other research on add-on projects
Other For pilot research purposes
Other molecular genetics
Statement 11: Beyond diagnosis and classification, diagnostic bone marrow aspirates should be used for LAIP identification, molecular MRD marker screening, as reference material for follow-up analyses, and biobanking.

8.3 Which pull of the bone marrow aspiration is used for MRD detection during follow-up?

8.4 If the first pull of bone marrow aspiration is not used for MRD assessment, what is it typically used for?

Statement 12: The first pull of bone marrow aspiration should be used for MRD assessment, as hemodilution in later pulls may hamper accurate MRD quantification.

8.5 What collection system do you use for bone marrow aspiration?

Response Answer
Other See Kiel SOP
Other Don’t know if it is standardized
Other do not know and I am afraid to go ask somewhere because it seems impossible to save the answers and I would loose too much work
Other systems differ between clinical departments

8.6 What is the volume of the collection system you use for bone marrow aspiration?

Response Answer
Other not applicable
Other 2-5 ml left and 2-5 ml right
Other depending of age and collection system all of the answers above

8.7 What volume of bone marrow do you typically aspirate during the procedure?

Response Answer
Other in multiple aspirates
Other Following the SOP , we perform 2 aspirates of 4ml (1st aspirate) and 10ml (2nd aspirate at different angle through same skin puncture)
Other we do repeat pulls of approximately 2ccs with reanchoring
Other depending of age and collection system all of the answers above

8.8 What volume of bone marrow do you typically provide for MRD analysis?

Category Comment
Other we receive DNA already extracted
Other Optimally 2.0 mL
Other depends on the cell number
6–10 mL depending of age and collection system all of the answers above
Statement 13: Up to 10mL of bone marrow should be aspirated in the first pull, of which 2–5mL should be provided for MRD analysis.

9 Trephine biopsy

9.1 Do you perform bone marrow trephine biopsy?

9.2 What is your typical approach in cases of a dry tap during bone marrow aspiration?

Statement 14: In the case of a dry tap during bone marrow aspiration, a trephine biopsy should be performed.
Statement 15: In the case of a dry tap during bone marrow aspiration, a second aspiration should be attempted at a different site.

10 Transport times

10.1 What is the typical transport time for bone marrow samples used for molecular MRD analysis?

Statement 16: The transport time for bone marrow samples intended for molecular MRD analysis should not exceed 48 hours.

10.2 What is the typical transport time for bone marrow samples used in flow cytometry MRD analysis?

Statement 17: The transport time for bone marrow samples intended for flow cytometry MRD analysis should not exceed 12 hours.
Statement 18: The transport time for bone marrow samples intended for flow cytometry MRD analysis should not exceed 48 hours.

11 Sample preparation

11.1 Are your bone marrow samples being checked for hemodilution?

Category Comment
No, never We send the sample to the central lab for FCM and there check for hemodilution
Yes, using Cytomorphology Presence/absence of bone marrow particles
Yes, using Cytomorphology assessment of spicules
Yes, using Cytomorphology Important for remission evaluation at day 33 (TP1)
Yes, using Flow cytometry checked for but no calculation, remark made in conclusion
Yes, using Flow cytometry do not know and do not want to go ask FC guys because it seems impossible to save the answers in the process. can send later
Yes, using Flow cytometry Presence of <2% of EBL
Yes, using Flow cytometry Only day 15: Important for FCM-MRD evaluation

11.2 If your bone marrow samples are assessed for hemodilution, how do you utilize the obtained information?

  • only remark with warning in conclusion
  • The test is performed anyway, final evaluation with the physician
  • A new sample is performed in case of hemodilution
  • write a comment to the results
  • If high-risk criteria are fulfilled (depending on the TP), resuts will be released for clinical used but withhold by the lab below those limits.
  • A comment is detailed in the cytomorphology report.
  • We might repeat depending on how much the result impacts clinical care.
  • Generally still used but with note made of hemodilution
  • Hemodilution is mostly relevant fror Response Evaluation at day 33 (TP1). If cytomorphology showes signs of heodilution and therefore a not representative BM, the BM puncture should be repeated. However we always measure MRD in both samples and use the higher value for stratification, because unsually MRD-levels are decreasing even without continuation of therapy after Day 33.
Statement 19: Bone marrow samples should be checked for hemodilution using either cytomorphology or flow cytometry; if hemodilution is detected, it should be noted in the report.

11.3 Are your bone marrow samples being processed using Ficoll separation prior to MRD analysis?

Statement 20: Bone marrow samples should be processed using Ficoll separation prior to molecular MRD analysis.

11.4 When preparing the first dilution step (typically 1E-01) for the standard curve from a Ficoll-processed sample, do you take the blast percentage into account?

Category Comment
Other yes, when blasts are below 50%
Other yes if it is below 50%
Other this varies based on each center practice
Other we adjust the standard curve according to cytomorphology, if blast infiltration is below 50% however we are trying to implement Flow after Ficoll at the moment in order to be more exact
Statement 21: When preparing the first dilution step (typically 1×10⁻¹) for the standard curve from a Ficoll-processed sample, the blast percentage should always be taken into account.

11.5 Is flow cytometry analysis performed before or after Ficoll separation?

Category Comment
Other I do not know
Other and after for defining blast percentage for molecular standard curve
Other before, in some situations (low number of blasts, sorting for RNAseq) later
Other The complete Flow cytometry analysis is performed before Ficoll separation. After Ficoll separation we perform an additional tube to calculate the blast percentage post-Ficoll
Other planned: after ficoll
Statement 22: Flow cytometry MRD analysis should be performed before Ficoll separation.
Statement 23: Flow cytometry MRD analysis should be performed after Ficoll separation.

11.6 Is cytomorphology analysis performed before or after Ficoll separation?

Category Comment
Other not performed
Other cytomorphology is performed on BM smears and not in the same sample
Statement 24: Cytomorphology should be performed before Ficoll separation.

12 MRD assesment methods

12.1 Which method(s) do you employ for MRD assesment in Ph-neg, KMT2A-neg BCP-ALL?

12.2 Which method(s) do you employ for MRD assesment in Ph-positive BCP-ALL?

12.3 Which method(s) do you employ for MRD assesment in KMT2A-positive BCP-ALL?

12.4 Which method(s) do you employ for MRD assesment in T-ALL?

12.5 Do you use any molecular markers for MRD analysis other than those mentioned above?

Category Comment
Other depends on target, number of classical targets
Other no, not at the moment
Other If flow unavailable
Other SIL-TAL and IKZf1

12.6 Which additional molecular markers do you use for MRD analysis?

13 Technical requirements flow cytometry

13.1 Which anticoagulant do you use for bone marrow samples intended for flow-cytometry MRD analysis?

Statement 25: Bone marrow samples intended for flow cytometry MRD analysis should be collected using either heparin or EDTA as an anticoagulant.

13.2 Do you apply bulk lysis to your samples?

Category Comment
NA I do not know
Only at certain time points (please specify) Not in aplasia
Only at certain time points (please specify) in case of low cellularity
NA Sometimes
Only at certain time points (please specify) only remission samples (day 33 onwards), not day 15 and not inital diagnosis
Statement 26: Bulk lysis should be applied to bone marrow samples prior to flow cytometry analysis.

13.3 What is the minimum number of cells you typically measure?

Category Comment
Other I do not know ( I am clinician)
Other Depends on the time-point. In ALL-BFM, we do day 15 MRD, and there we acquire 500,000 cells. In GMALL and in ALL-BFM at other time-points, we aim for 4,000,000 events.
Other Dx 100,000, day 15 100,000-500,000, day 33 more than 1,000,000
Other 500,000 cells
Other Varies based on lab and method
Other remission spamples:1-10. Mio, Initial + day 15 : 100.000-500,000

13.4 What is the minimum number of events required to define MRD positivity (LOD)?

Category Comment
Other do not know ( I am clinician)
Other 10 events
Other Varies based on lab and method
Statement 27: A minimum of 10–20 events is required to define MRD positivity (limit of detection) in flow cytometry analysis.

13.5 What is the minimum number of events required to quantify MRD (LOQ)?

Category Comment
NA do not know ( I am clinician)
Other 40 events
Other Varies based on lab and method
Statement 28: A minimum of 10–20 events is required to quantify MRD (limit of quantification) in flow cytometry analysis.
Statement 29: A minimum of 21-50 events is required to quantify MRD (limit of quantification) in flow cytometry analysis.

13.6 What level of sensitivity (LOD) is typically achieved in your MRD analysis?

Response Answer
Other BCP-ALL: E-05, T-ALL: rather E-04 or less
Other depends on timepoint, see above
Other 2X10-5
Other It varies according to the time point
Other day 15: 1E-03 (without bulk lyisis)
Statement 30: Flow cytometry MRD analysis should achieve a sensitivity of at least 1×10⁻⁴
Statement 31: Flow cytometry MRD analysis should achieve a sensitivity of at least 5×10⁻⁵
Statement 32: Flow cytometry MRD analysis should achieve a sensitivity of at least 1×10⁻⁵
Statement 33: Flow cytometry MRD analysis should achieve a sensitivity of at least 1×10⁻⁶

13.7 What level of quantification (LOQ) is typically achieved in your MRD analysis?

Response Answer
Other BCP-ALL: 5xE-05, T-ALL: rather E-04 or less
Other day 15 0.1%, day 33 0.1%-0.01%
Other 6X10-5
Other It varies according to the time point
Statement 34: Flow cytometry MRD analysis should achieve a limit of quantification of at least 1×10⁻⁴
Statement 35: Flow cytometry MRD analysis should achieve a limit of quantification of at least 5×10⁻⁵
Statement 36: Flow cytometry MRD analysis should achieve a limit of quantification of at least 1×10⁻⁵
Statement 37: Flow cytometry MRD analysis should achieve a limit of quantification of at least 1×10⁻⁶

13.8 Do you use a commercial assay or an in-house assay for your MRD analysis?

Category Comment
Commercial NSG MRD is commercial
Commercial ClonoSeq for NGS-based
EuroFlow BCP-ALL: EuroFlow, but as in-house assay. T-ALL: in-house assay according to BFM-Flow SOP
EuroFlow BCP-ALL MRD assay
EuroFlow BD CytognosTM B-cell Precursors ALL MRD
EuroFlow B-MRD plus 1 additional in-house tube
In-house I-BFM standard
In-house Flow is both performed locally and centrally and depends on population and trial and timepoints
In-house St. Jude in house
In-house T-MRD

14 Technical requirements IG/TR qPCR

14.1 Which anticoagulant do you use for bone marrow samples intended for IG/TR-based qPCR MRD analysis?

14.2 What is the minimum DNA amount you typically analyze per reaction?

## No comments available.

14.3 What level of sensitivity is typically achieved in your MRD analysis?

14.4 What quantitative range is typically achieved in your MRD analysis?

14.5 How do you report results that are above the sensitivity threshold but below the quantitative range?

15 Technical Requirements IG/TR ddPCR

15.1 Which anticoagulant do you use for bone marrow samples intended for IG/TR-based ddPCR MRD analysis?

15.2 What is the minimum DNA amount you typically analyze per molecular target?

## No comments available.

15.3 What is the minimum number of events required to define MRD positivity?

## No comments available.

15.4 What level of sensitivity is typically achieved in your MRD analysis?

15.5 Do you account for background noise from healthy cells in your MRD analysis?

15.6 Do you use a commercial assay or an in-house assay for your MRD analysis?

## No comments available.

16 Technical Requirements IG/TR amplicon NGS

16.1 Which anticoagulant do you use for bone marrow samples intended for IG/TR-based NGS MRD analysis?

16.2 What is the minimum DNA amount you typically analyze per sequencing library reacion?

Category Comment
Other This is centrally done and we are not privy to the amount needed

16.3 What is the minimum number of reads required to define MRD positivity?

Category Comment
Other This is proprietary and we are not provided it

16.4 What level of sensitivity is typically achieved in your MRD analysis?

16.5 What quantitative range is typically achieved in your MRD analysis?

16.6 Do you account for background noise from healthy cells in your MRD analysis?

16.7 Do you use a commercial assay or an in-house assay for your MRD analysis?

Category Comment
Commercial clonoSEQ
Commercial Clonoseq

17 Technical requirements KMT2A qPCR

17.1 Which anticoagulant do you use for bone marrow samples intended for KMT2A-based qPCR MRD analysis?

17.2 What is the minimum DNA amount you typically analyze per reaction?

## No comments available.

17.3 What level of sensitivity is typically achieved in your MRD analysis?

17.4 What quantitative range is typically achieved in your MRD analysis?

17.5 Do you account for background noise from healthy cells in your MRD analysis?

17.6 Do you use a commercial assay or an in-house assay for your MRD analysis?

Category Comment
In-house RT-PCR; note, we use RNA rather than DNA
In-house patient-specific assays

18 Technical Requirements KMT2A ddPCR

no responses

19 Technical Requirements DNA-based BCR::ABL1 qPCR

19.1 Which anticoagulant do you use for bone marrow samples intended for BCR::ABL1-based qPCR MRD analysis?

19.2 Which housekeeping gene do you use for BCR::ABL1 qPCR normalization?

19.3 How many copies of the housekeeping gene do you typically aim for in BCR::ABL1 transcript quantification?

Category Comment
Other depends on situation (we use GUSB)
NA not applicable for DNA-based analysis

19.4 How do you quantify the housekeeping gene as the denominator for BCR::ABL1 MRD assessment:

Category Comment
NA not applicable for DNA-based analysis

19.5 What is the source of the plasmids you use for BCR::ABL1 quantification?

19.6 What is the minimum number of positive replicates required to define MRD positivity?

Category Comment
NA not applicable for DNA-based analysis

19.7 How do you report the MRD value for the major BCR::ABL1 transcripts?

19.8 How do you report the MRD value for the minor BCR::ABL1 transcripts?

19.9 Do you use the CML MRD nomenclature to interpret BCR::ABL1-MRD results in Ph+ ALL (MMR, MR4, MR4.5, MR5)?

Category Comment
NA not applicable for DNA-based analysis

19.10 Do you use a commercial assay or an in-house assay for major BCR::ABL1-transcript-based MRD analysis?

Category Comment
In-house patient-specific assay

19.11 Do you use a commercial assay or an in-house assay for minor BCR::ABL1-transcript-based MRD analysis?

Category Comment
In-house patient-specific assay

20 Technical Requirements RNA-based BCR::ABL1 qPCR

20.1 Which anticoagulant do you use for bone marrow samples intended for BCR::ABL1-based qPCR MRD analysis?

20.2 Which housekeeping gene do you use for BCR::ABL1 qPCR normalization?

20.3 How many copies of the housekeeping gene do you typically aim for in BCR::ABL1 transcript quantification?

Category Comment
NA do not know ( I am clinician)
Other aim for >100,000 ABL1 copies
Other NA (Cepheid)
Other depends on situation, in poor samples we use bad quality samples and comment
Other 15-35 copies GAPDH

20.4 How do you quantify the housekeeping gene as the denominator for BCR::ABL1 MRD assessment:

Category Comment
NA do not know ( I am clinician)
Other cepheid

20.5 What is the source of the plasmids you use for BCR::ABL1 quantification?

20.6 How do you report the MRD value for the major BCR::ABL1 transcripts?

20.7 What is the minimum number of positive replicates required to define MRD positivity?

Category Comment
NA do not know ( I am clinician)

20.8 How do you report the MRD value for the minor BCR::ABL1 transcripts?

20.9 Do you use the CML MRD nomenclature to interpret BCR::ABL1-MRD results in Ph+ ALL (MMR, MR4, MR4.5, MR5)?

Category Comment
NA do not know ( I am clinician)
Other Only if a clinical trial asks us to do so

20.10 Do you use a commercial assay or an in-house assay for major BCR::ABL1-transcript-based MRD analysis?

Category Comment
Commercial Cepheid
Commercial Qiagene
Commercial Xpert BCR-ABL ULTRA (Cepheid)
Commercial commercial for CML
In-house IRMM ERM-AD623 Plasmid and in-house assay
In-house in house for MRD in Ph+ ALL

20.11 Do you use a commercial assay or an in-house assay for minor BCR::ABL1-transcript-based MRD analysis?

Category Comment
Commercial Ipsogen BCR::ABL1 m-bcr Kit
Commercial Cepheid
Commercial Qiagene
Commercial commercial for CML
In-house in house for MRD in Ph+ ALL

21 Technical Requirements DNA-based BCR::ABL1 ddPCR

no responses

22 Technical Requirements RNA-based BCR::ABL1 ddPCR

no responses

23 Technical requirements STIL::TAL1 qPCR

23.1 Which anticoagulant do you use for bone marrow samples intended for STIL::TAL1-based qPCR MRD analysis?

23.2 What is the minimum DNA amount you typically analyze?

## No comments available.

23.3 What level of sensitivity is typically achieved in your MRD analysis?

23.4 What quantitative range is typically achieved in your MRD analysis?

23.5 Do you account for background noise from healthy cells in your MRD analysis?

23.6 Do you use a commercial assay or an in-house assay for your MRD analysis?

## No comments available.

24 Technical requirements STIL::TAL1 ddPCR

no responses

25 Molecular MRD markers

25.1 How do you manage patients who do not have a suitable molecular MRD marker?

26 Prioritization in cases with multiple MRD results

26.1 If both BCR::ABL1-based MRD on a DNA-level and IG/TR-based MRD results are available, which one do you prioritize for reporting?

Category Comment
Other do not use DNA for BCR-ABL
Other only IG/TR performed
Other We do not perform BCR::ABL1-based DNA MRD. However, in case such result was available, both results would be reported from the lab to the clinicians. The concordance or discordance of results and final conclusions are discussed in a multidisciplinary meeting for every MRD assessment for all patients
Other do not use IG/TR-MRD
Other BCR::ABL1 is not used for stratification

26.2 If both BCR::ABL1-based MRD on an RNA-level and IG/TR-based MRD results are available, which one do you prioritize for reporting?

Category Comment
Other Both results are reported from the lab to the clinicians. The concordance or discordance of results and final conclusions are discussed in a multidisciplinary meeting for every MRD assessment for all patients. However, in the EsPhALL trial only the IG-TR MRD results are considered.
Other do not use IG/TR-MRD
IG/TR-based MRD result BCR::ABL1 is not used for stratification

26.3 If both KMT2A-based MRD and IG/TR-based MRD results are available, which one do you prioritize for reporting?

Category Comment
Other We do not perform KMT2A-based MRD. However, in case such result was available, both results would be reported from the lab to the clinicians. The concordance or discordance of results and final conclusions are discussed in a multidisciplinary meeting for every MRD assessment for all patients. Generally speaking, we would consider the higher MRD value
Other do not use IG/TR-MRD

26.4 In switch ALL cases, which MRD method do you prioritize for reporting?

Category Comment
Other both results with comment from FC group
Other Both results are reported from the lab to the clinicians. The concordance or discordance of results and final conclusions are discussed in a multidisciplinary meeting for every MRD assessment for all patients. In switch ALL cases, we usually find higher MRD values with IG/TR and consider this higher MRD result for taking clinical actions.
Other in the US all results have to be reported if a clinical test. Protocols can give guidance and local institutions have their own standard. But it is not allowed for the lab who performs the assay to prioritize

27 MRD vs. cytomorphology for remission assessment

MRD and cytomorphology results are not always concordant. In the following situations, which value do you choose for clinical reporting to state the blast percentage at the time of remission assessment?

27.1 Cytomorphology >25% and:

27.1.1 MRD <1%

27.1.2 MRD 1-5%

27.1.3 MRD 5-25%

27.1.4 MRD >25%

27.2 Cytomorphology 5-25% and:

27.2.1 MRD <1%

27.2.2 MRD 1-5%

27.2.3 MRD 5-25%

27.2.4 MRD >25%

27.3 Cytomorphology <5% and:

27.3.1 MRD <1%

27.3.2 MRD 1-5%

27.3.3 MRD 5-25%

27.3.4 MRD >25%

28 MRD vs. cytomorphology for relapse assessment

MRD and cytomorphology results are not always concordant. In the following situations, which value do you choose for clinical reporting to state the blast percentage at the time of relapse assessment?

28.1 Cytomorphology >25% and:

28.1.1 MRD <1%

28.1.2 MRD 1-5%

28.1.3 MRD 5-25%

28.1.4 MRD >25%

28.2 Cytomorphology 5-25% and:

28.2.1 MRD <1%

28.2.2 MRD 1-5%

28.2.3 MRD 5-25%

28.2.4 MRD >25%

28.3 Cytomorphology <5% and:

28.3.1 MRD <1%

28.3.2 MRD 1-5%

28.3.3 MRD 5-25%

28.3.4 MRD >25%

29 CSF MDD

29.1 Do you perform MDD assessment in cerebrospinal fluid?

Category Comment
Other we sometimes receive CSF, not systematically recommended
Other if asked and sent by clinicians
Other rarely to confirm antigen expression following identified CNS involvement
Other only in case of suspected relapse

29.2 If MDD is detected in cerebrospinal fluid, are there any therapeutic consequences?

Category Comment
Other only CNS3
Other No, but performing flow-cytometry assessment is strongly recommended by the protocol to confirm the presence of blasts identified by cytomorphology
Other not sure, not standardized

30 CSF MRD

30.1 Do you perform MRD assessment in cerebrospinal fluid?

Category Comment
Other we sometimes receive SCF, but it is not systematically recommended
Other if asked and sent by clinicians
Other not sure, not standardized
Other rarely to confirm antigen expression following identified CNS involvement
Other only in case of suspected CNS-relapse/persisting blasts in cytospin

30.2 If MRD is detected in cerebrospinal fluid, are there any therapeutic consequences?

Category Comment
Other if MRD+ switch of therapy
Other Each assessment is discussed in a joint meeting of the laboratories and the clinical teams

31 CSF MRD Methods

31.1 Which method do you use for MRD assessment in cerebrospinal fluid?

31.2 How is MRD positivity in cerebrospinal fluid defined when detected using IG/TR qPCR?

Category Comment
Other (please comment) We do not use IG/TR qPCR
Other (please comment) We do not perform IG/TR MRD in CSF
According to CSF-specific rules (please comment) not standardized yet

31.3 What is the minimum number of events required to define MRD positivity in cerebrospinal fluid by flow cytometry?

Category Comment
NA do not know ( I am clinician)

32 Additional topics

32.1 In your opinion, are there any important topics we have missed in this survey? If yes, please specify in the comments.

Category Comment
No I cannot successfully answer to many technical comments, as I am a clinician
Yes some questions are difficult to answer, this answers would be much more complicated.
No level of control gene in DNA-based methods (which is acceptable)-there are transcript copies.
Yes Communication between the different labs and between the lab and the clinical teams is crucial to discuss both the concordant and the discordant results. We perform an integrated biological report at diagnosis and at each MRD time point, and each BM assessment is discussed weekly with the clinicians.

A possible topic to discuss is the presence of integrated reports and the (fluid)communication between clinicians and laboratory staff.

Comment for questions 148-153: both the cytomorphology and the MRD results are reported from the lab, and any discordances are studied and discussed in a meeting with the lab and clinical teams to try to explain the discordance (hemodilution of the sample, regenerative BM with a high percentage of B-cell normal precursors (hematogonies), difficult LAIPs, etc) and agree on a conclusion on the results. | |Yes |planned / expected changed in the near future | |Yes |diagnostic procedures and laboratory practices will be country specific, in the UK we operate via the SIHMDS model. For instance, it is not our practice to regress or censor multimodal results for remission to one output, all our reported out to the requesting clinician. In addition practices are guided by the treatment approval criteria - e.g. flow based assessments for CAR-T indication but molecular MRD (plus flow) for Blina for instance, so no singular system exists addressing all the clinically actionable outcomes of MRD testing |

33 All statements for delphi poll

Statement # Statement
Statement 1 MRD thresholds below 1×10⁻⁴ are not relevant for risk stratification in frontline treatment of ALL.
Statement 2 Bone marrow aspiration should be performed in all patients for postremission MRD monitoring to enable early detection of relapse.
Statement 3 Postremission MRD assessment should be performed at fixed intervals according to the protocol and additionally in response to clinical signs of relapse.
Statement 4 Bone marrow aspiration for relapse detection should be continued until 36–60 months after initial diagnosis.
Statement 5 Bone marrow aspiration for relapse detection should be continued until 36 months after stem cell transplantation.
Statement 6 For postremission relapse detection, any MRD positivity is considered relevant; however, values below 1×10⁻⁴ should be confirmed using an additional method and a second sample.
Statement 7 Conversion to MRD positivity should be considered a relapse only if the MRD level exceeds 1×10⁻⁴ and is confirmed by an additional method or other molecular target.
Statement 8 Conversion from IG/TR-based MRD-negativity to MRD-positivity should be confirmed by a consecutive sample only if the MRD level is below 1×10⁻⁴.
Statement 9 Conversion from BCR::ABL1-based MRD-negativity to MRD-positivity should be considered a relapse only if accompanied by IG/TR-based MRD positivity.
Statement 10 If MRD conversion requires confirmation, the consecutive sample should be taken within 1–4 weeks.
Statement 11 Beyond diagnosis and classification, diagnostic bone marrow aspirates should be used for LAIP identification, molecular MRD marker screening, as reference material for follow-up analyses, and biobanking.
Statement 12 The first pull of bone marrow aspiration should be used for MRD assessment, as hemodilution in later pulls may hamper accurate MRD quantification.
Statement 13 Up to 10mL of bone marrow should be aspirated in the first pull, of which 2–5mL should be provided for MRD analysis.
Statement 14 In the case of a dry tap during bone marrow aspiration, a trephine biopsy should be performed.
Statement 15 In the case of a dry tap during bone marrow aspiration, a second aspiration should be attempted at a different site.
Statement 16 The transport time for bone marrow samples intended for molecular MRD analysis should not exceed 48 hours.
Statement 17 The transport time for bone marrow samples intended for flow cytometry MRD analysis should not exceed 12 hours.
Statement 18 The transport time for bone marrow samples intended for flow cytometry MRD analysis should not exceed 48 hours.
Statement 19 Bone marrow samples should be checked for hemodilution using either cytomorphology or flow cytometry; if hemodilution is detected, it should be noted in the report.
Statement 20 Bone marrow samples should be processed using Ficoll separation prior to molecular MRD analysis.
Statement 21 When preparing the first dilution step (typically 1×10⁻¹) for the standard curve from a Ficoll-processed sample, the blast percentage should always be taken into account.
Statement 22 Flow cytometry MRD analysis should be performed before Ficoll separation.
Statement 23 Flow cytometry MRD analysis should be performed after Ficoll separation.
Statement 24 Cytomorphology should be performed before Ficoll separation.
Statement 25 Bone marrow samples intended for flow cytometry MRD analysis should be collected using either heparin or EDTA as an anticoagulant.
Statement 26 Bulk lysis should be applied to bone marrow samples prior to flow cytometry analysis.
Statement 27 A minimum of 10–20 events is required to define MRD positivity (limit of detection) in flow cytometry analysis.
Statement 28 A minimum of 10–20 events is required to quantify MRD (limit of quantification) in flow cytometry analysis.
Statement 29 A minimum of 21-50 events is required to quantify MRD (limit of quantification) in flow cytometry analysis.
Statement 30 Flow cytometry MRD analysis should achieve a sensitivity of at least 1×10⁻⁴
Statement 31 Flow cytometry MRD analysis should achieve a sensitivity of at least 5×10⁻⁵
Statement 32 Flow cytometry MRD analysis should achieve a sensitivity of at least 1×10⁻⁵
Statement 33 Flow cytometry MRD analysis should achieve a sensitivity of at least 1×10⁻⁶
Statement 34 Flow cytometry MRD analysis should achieve a limit of quantification of at least 1×10⁻⁴
Statement 35 Flow cytometry MRD analysis should achieve a limit of quantification of at least 5×10⁻⁵
Statement 36 Flow cytometry MRD analysis should achieve a limit of quantification of at least 1×10⁻⁵
Statement 37 Flow cytometry MRD analysis should achieve a limit of quantification of at least 1×10⁻⁶

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